Amyotrophic lateral sclerosis (ALS) is an almost invariably lethal disorder which involves loss of the large motor neurons from the cerebral cortex, brain stem, and spinal cord. During the past three decades, mortality due to ALS has increased consistently. While most cases of ALS are sporadic, 5-10% of cases (which are clinically indistinguishable) are familial. Recently, we have identified the gene responsible for ALS in a subset of the familial cases: Cu/Zn superoxide dismutase (SOD-1), an enzyme which inactivates the highly reactive free radical superoxide anion. The aim of this study is to establish and characterize an in vitro model of ALS, in order to identify the mechanisms by which mutations in the SOD-1 gene lead to the death of motor neurons. We propose to study this by the preparation and characterization of motor neurons which are stably transduced with human SOD-1 cDNA containing one of eleven different mutations identified in our FALS patients. We aim to: 1) Prepare constructs with human SOD-1 cDNAs, both control and mutants. 2) Package mutant SOD-1 constructs into Adeno-Associated Virus (AAV) Vectors. AAV is a human DNA virus which offers a promising alternative to the viral vectors now in frequent use (adenovirus, Herpes, retroviruses). 3) Prepare cultures of rat motor neurons grown under defined conditions which will be characterized for survival, morphology, neurite outgrowth, and expression of neuronal markers. 4) Transduce cultured motor neurons with mutant-human SOD-1 using a replication deficient AAV vector. 5) Measure the activity of enzymes responsible for the detoxification of free-radical metabolites such as superoxide anion and hydrogen peroxide, in control and transduced neurons. And, 6) Use infected motor neurons to test the effectiveness of several therapeutic approaches: trophic factors such as NT-3, BDNF, or NT 4/5, that have motor neurons as targets; pharmacologic agents which are believed to counteract the effects of excessive free radicals; or genes, such as bcl-2, which have been shown to rescue cells from death. Therapeutic agents can be introduced directly into the culture medium (pharmaceutics, trophic factors); secreted by co-cultured, genetically engineered astrocytes (trophic factors); or introduced with the human SOD- 1 cDNA by transduction of a bicistronic AAV construct (trophic factors, cell survival genes). The long-term objective of this project is to identify putative therapeutic agents that will prolong the survival of diseased or injured motor neurons.